Selective degradation of the p53‐R175H oncogenic hotspot mutant by an RNA aptamer‐based PROTAC

To the Editor: TP53 encodes the tumour suppressor protein p53, a master regulator of genomic integrity and cell survival, and is the most frequently mutated gene.1 p53 mutant proteins are stabilised and can acquire dominant-negative or oncogenic gain-of-function activities, thereby promotingmalignant transformation, metastasis and chemoresistance.2,3 Proteolysis targeting chimeras (PROTACs) that hijack cellular ubiquitin-proteasomemachinery for targeted protein degradation have shown considerable promise in targeting previously undruggable proteins.4,5 However, PROTACs targeting p53mutants have not yet been reported, probably due to difficulty in identifying a suitable binder for these mutants. Some recent studies have revealed that aptamers can be exploited as ligands in place of standard peptides or small molecules for PROTACs.6–8 Here, using an aptamerbased strategy, we developed the first selective hotspot p53 mutant PROTAC. p53-R175H is the most common p53 hotspot mutation.9 In this study, we used an RNA aptamer10 (hereafter named p53m-RA) that selectively targets p53-R175H as a binder for PROTACdevelopment. First, we used streptavidin pulldown assays to confirm its binding specificity. Both p53mRA and N3-p53m-RA competitively abolished p53-R175H pulldown from cell lysates with N3-p53m-RA-biotin. By contrast, there was almost no pulldown of wild-type p53 (p53-WT) (Figure 1A). Structural analysis revealed that p53-R175H displays a much narrower binding area for the DNA major groove than the wild-type p53 (Figure 1B), but the newly formed groove between L2 and L3 facilitates the binding of p53m-RA to p53-R175H (Figure 1C). Moreover, the 5′ end of p53m-RA was far away from the ligand binding sites (Figure 1D). Therefore, an alkynylated CRBN ligand (CRBNL), thalidomide-O-amido-propargyl (Supporting Information Schemes), was connected to the 5′ end of N3-p53m-RA via a click reaction to generate the p53-R175H degrader, dp53m-RA (Figure 1E).

To the Editor: TP53 encodes the tumour suppressor protein p53, a master regulator of genomic integrity and cell survival, and is the most frequently mutated gene. 1 p53 mutant proteins are stabilised and can acquire dominant-negative or oncogenic gain-of-function activities, thereby promoting malignant transformation, metastasis and chemoresistance. 2,3 Proteolysis targeting chimeras (PROTACs) that hijack cellular ubiquitin-proteasome machinery for targeted protein degradation have shown considerable promise in targeting previously undruggable proteins. 4,5 However, PROTACs targeting p53 mutants have not yet been reported, probably due to difficulty in identifying a suitable binder for these mutants. Some recent studies have revealed that aptamers can be exploited as ligands in place of standard peptides or small molecules for PROTACs. [6][7][8] Here, using an aptamerbased strategy, we developed the first selective hotspot p53 mutant PROTAC.
significantly attenuated the migration of p53-R175Hexpressing cells, but not p53-WT-expressing cells ( Figure 3E-H). These results indicate that dp53m-RA may have therapeutic potential in patients harbouring p53-R175H mutant cancers.
In summary, by harnessing an RNA aptamer specifically targeting p53-R175H, the most common TP53 mutation with dominant-negative and oncogenic gain-offunction activities, as a binder in PROTAC design, we report the first selective mutant p53 degrader, dp53m-RA. showing the selective degradation of p53-R175H protein by dp53m-RA and its biological consequences. ***p < 0.001; Student's t-test; n.s.: not significant dp53m-RA degraded p53-R175H but not wild-type p53 or other p53 mutants in a ubiquitin-proteasome-dependent manner. Importantly, dp53m-RA inhibited the proliferation and migration of cancer cells specifically harbouring the p53-R175H mutation ( Figure 3I), suggesting its therapeutic potential for precision medicine.

A C K N O W L E D G E M E N T S
This work was supported by grants from the National Natural Science Foundation of China (32070708, 32270892, 81702678).

C O N F L I C T O F I N T E R E S T
The authors declare they have no conflicts of interest.